Electrochemical strategy for ultrasensitive detection of microRNA based on MNAzyme-mediated rolling circle amplification on a gold electrode

The authors describe an electrochemical strategy for ultrasensitive and specific detection of microRNA (miRNA). It is based on both multicomponent nucleic acid enzyme (MNAzyme) amplification and rolling circle amplification (RCA). In the presence of target miRNAs, partial enzyme A (partzyme A) and partial enzyme B (partzyme B) are assembled to form active MNAzymes. Once formed, the MNAzymes catalyze the cleavage of the hairpin substrates to liberate biotinylated fragments which hybridized with the capture probes immobilized on a gold electrode. The RCA is then initiated to form a product that binds many detection probes. Finally, the amperometric signal (best acquired at a working voltage of 0.22 V vs. Ag/AgCl) is obtained by employing the streptavidinylated alkaline phosphatase as the enzyme. This biosensor has a 1.66 fM detection limit, and a dynamic range that extends from 10 fM to 1 nM. It displays specificity down to single mismatch discrimination of target miRNAs and good reproducibility. It was successfully applied to the determination of miRNA in total RNA samples extracted from human breast adenocarcinoma MCF-7 cells.